Ras-induced epigenetic inactivation of the RRAD (Ras-related associated with diabetes) gene promotes glucose uptake in a human ovarian cancer model

Abstract

RRAD (Ras-related associated with diabetes) is a small Ras-related GTPase that is frequently inactivated by DNA methylation of the CpG island in its promoter region in cancer tissues. However, the role of the methylation-induced RRAD inactivation in tumorigenesis remains unclear. In this study, the Ras-regulated transcriptome and epigenome were profiled by comparing T29H (a Ras(V12)-transformed human ovarian epithelial cell line) with T29 (an immortalized but non-transformed cell line) through reduced representation bisulfite sequencing and digital gene expression. We found that Ras(V12)-mediated oncogenic transformation was accompanied by RRAD promoter hypermethylation and a concomitant loss of RRAD expression. In addition, we found that the RRAD promoter was hypermethylated, and its transcription was reduced in ovarian cancer versus normal ovarian tissues. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in demethylation in the RRAD promoter and restored RRAD expression in T29H cells. Additionally, treatment with farnesyltransferase inhibitor FTI277 resulted in restored RRAD expression and inhibited DNA methytransferase expression and activity in T29H cells. By employing knockdown and overexpression techniques in T29 and T29H, respectively, we found that RRAD inhibited glucose uptake and lactate production by repressing the expression of glucose transporters. Finally, RRAD overexpression in T29H cells inhibited tumor formation in nude mice, suggesting that RRAD is a tumor suppressor gene. Our results indicate that Ras(V12)-mediated oncogenic transformation induces RRAD epigenetic inactivation, which in turn promotes glucose uptake and may contribute to ovarian cancer tumorigenesis.

Keywords: DNA Methylation; Metabolism; Oncogenic Transformation; Ovarian Cancer; RRAD; Ras; Tumor Suppressor Gene.

FIGURE 1.

RRAD DNA methylation is inversely correlated with expression in cultured ovarian epithelial cells. A, starburst plot showing the association between DMRs in the promoter and the gene expression level. B, detailed view of DMR of RRAD from the RRBS-seq data. C, DNA methylation status of the RRAD promoter in T29 and T29H cells (***, p < 0.001). D, reduced RRAD mRNA levels in T29H versus T29 cells. E, reduced RRAD protein levels in T29H cells. Data are shown as means ± S.E. (error bars) (**, p < 0.01); β-actin was used as an internal control.

FIGURE 2.

RRAD promoter methylation is inversely correlated with expression in epithelial ovarian carcinoma. A, methylation status of the RRAD promoter region in normal tissues and epithelial ovarian carcinoma. Each filled bar represents the percentage of DNA methylation per CpG site, and the open bar represents the percentage of demethylated CpG at each corresponding site (**, p = 0.0045). B, overall comparison of relative RRAD mRNA levels in ovarian cancer (n = 42) and normal tissues (n = 10). Data are shown as means ± S.E. (error bars) (*, p < 0.05, Mann-Whitney U test). C, RRAD mRNA levels in individual cancer tissues and the reference tissue sample, which was mixed from 10 normal tissues.

FIGURE 3.

Demethylation of RRAD in T29H by 5-aza-dC results in RRAD gene derepression. A, DNA methylation status of the RRAD promoter in 5-aza-dC-treated T29H cells. The red box marks the “hot spots” that are demethylated upon 5-aza-dC treatment (*, p = 0.027). B, relative RRAD mRNA levels after 5-aza-dC treatment. C, RRAD protein levels after treatment with 20 μm 5-aza-dC. D, DNMT mRNA levels in T29H. E, relative DNMT mRNA levels after FTI277 treatment. F, relative DNMT activity following FTI277 treatment. G, relative mRNA levels of RRAD following FTI277 treatment. β-Actin was used as a loading control. Data are shown as means ± S.E. (error bars) (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 4.

RRAD expression is regulated by HRas^V12. A–C, overexpression of HRas^V12 in T29 cells. A, HRas^V12 transfection efficiency in T29 cells, measured by real-time RT-PCR. B and C, reduced protein (B) and mRNA (C) levels of RRAD in HRas^V12-overexpressing T29 cells. D–F, siRNA knockdown of HRas^V12 in T29H cells. D, transfection efficiency of HRas^V12 siRNA in T29H cells. E and F, up-regulation of RRAD in HRas^V12 knockdown T29H cells. E, protein level; F, relative RRAD transcript level. Data are shown as means ± S.E. (error bars) (**, p < 0.01).

FIGURE 5.

RRAD inhibits glucose uptake and lactate production in ovarian epithelial cells. A, reduced glucose uptake and lactate production in T29 cells versus T29H cells that express lower levels of RRAD. B–D, increased glucose uptake and lactate production in siRRAD knockdown T29 cells. B, relative glucose and lactate levels. C, relative RRAD transcript level. D, RRAD protein expression level. E–G, significantly decreased glucose uptake and lactate production upon RRAD overexpression in T29H cells. E, relative glucose and lactate levels. F, relative RRAD transcript level. G, RRAD protein expression level. Data are means ± S.E. (error bars) (*, p < 0.05; **, p < 0.01).

FIGURE 6.

Glucose transporter expression in T29, T29H, and RRAD-overexpressing T29H cells. A, T29H cells were transfected with pCMV6-XL5 RRAD or pcDNA3.1, and the levels of glucose transporters were determined by real-time RT-PCR 48 h after transfection. B, real-time RT-PCR quantification of glucose transporters in T29 and T29H cells. Data are means ± S.E. (error bars) (*, p < 0.05; **, p < 0.01).

FIGURE 7.

RRAD reduces ovarian tumor growth in vivo. A, relative RRAD transcript levels in T29H and its stable RRAD-overexpressing derivatives G7 and G8. B, RRAD protein expression in T29H and its stable RRAD-overexpressing derivatives. C, reduced lactate production in stable RRAD-overexpressing T29H derivatives. D, representative images of mice in T29/T29H mouse models 6 weeks after subcutaneous injection. T29 did not form visible tumors; however, mice injected with T29H, T29H-G7, T29H-G8 all formed visible tumors. Due to image quality, images of only 3 mice per treatment are shown. E, quantification of tumor volume from subcutaneous injected athymic nude mice, n = 5. F, representative images of autopsied tumors from T29H, G7, and G8 subcutaneously injected athymic nude mice. Data are means ± S.E. (error bars) (**, p < 0.01).

FIGURE 8.

Genomic landscape of DNA methylation in T29 and T29H determined by RRBS-seq. A, the overall methylated level of C and CG in T29 and T29H. B, global DNA methylation analysis of methylated level in T29 and T29H using the LC-MS/MS method.